阿拉伯芥麩胺酸受體3.6及麩胺酸參與根之生長發育調控

Regulation of Root Development by Glutamate Receptor-Like gene GLR3.6 and Glutamate in Arabidopsis thaliana

夏許(Shashi Kant Singh)

指導教授 ： 張英峯

國立臺灣大學/生命科學院/植物科學研究所/博士(2016年)

https://doi.org/10.6342/NTU201600219

摘要

阿拉伯芥公認的類麩氨酸受體 (AtGLR)基因似乎是植物生長發育過程中重要的調節者，然而大多數的類麩氨酸受體其基因功能尚未明瞭。阿拉伯芥類麩氨酸受體3.6 (AtGLR3.6)是阿拉伯芥20個類麩氨酸受體的其中一員，屬於第三群分支。我們探討AtGLR3.6與阿拉伯芥根部發展的關係。實驗結果顯示AtGLR3.6基因能在植物根部及地上部表達；並且AtGLR3.6蛋白被發現位於細胞膜上。而我們也發現，AtGLR3.6發生突變而失去功能的植株 (glr3.6-1)導致主根變短並且具有較少的側根。在阿拉伯芥中大量表現阿拉伯芥AtGLR3.6基因促進側根及主根以及莖的生長。在阿拉伯芥glr3.6-1突變株與野生株比較，顯示出較小的根的分生組織大小，說明阿拉伯芥AtGLR3.6控制根的分生組織大小。此外，阿拉伯芥glr3.6-1突變株表現出降低的有絲分裂活性，認為是跟減少根的分生組織大小有關。在阿拉伯芥glr3.6-1突變株的根中，細胞週期素依存性的激酶抑制劑Kip-related protein 4 (KRP4) 的表現量呈現正調控，而在阿拉伯芥AtGLR3.6大量表現株的根中則呈現負調控，意味著KRP4在經由阿拉伯芥AtGLR3.6調節的根的分生組織的維持扮演重要角色。在阿拉伯芥轉殖株中正調控KRP4，顯示出減少根和莖的生長，並且減少根的分生組織的大小。重要的是，當負調控KRP4基因表現時，glr3.6-1突變珠會回復大多數根的生長。然而，在阿拉伯芥AtGLR3.6 大量表現株當中提高KRP4表現量，會複製野生株根的生長。細胞質鈣離子濃度梯度在glr3.6-1突變株的根中會減少。再者，處理鈣離子減少KRP4 mRNA 程度，因此，誘導根的生長。我們使用一系列具有調節動物iGluR活性的化合物，為了進一步確認麩胺酸訊息傳遞的角色。我們使用麩胺酸及其他具有增加減少抗動物iGluR通道的化合物。iGluR受體的增加或減少改變阿拉伯芥根的生長發育。我們的結果證實增加iGluR促進根的生長並且減少iGluR大幅地抑制根的生長。在減少根處理的實驗中藉由額外添加麩胺酸使根的結構恢復。綜合以上所述，我們的發現指出阿拉伯芥麩胺酸受體3.6在側根和主根生長發育中是扮演一正向調節者。

關鍵字

麩胺酸受體 ； 側根 ； KRP4 ； AtGLR ； 生長素 ； 鈣離子

並列摘要

Arabidopsis putative glutamate receptor (AtGLR)-like genes seem important regulators participating in the plant growth and development processes, and the functions of the most of them remain unknown. AtGLR3.6 is one of the 20 AtGLR-like genes in Arabidopsis and belongs to clade-III. Here we have investigated the role of AtGLR3.6 in root development in Arabidopsis thaliana. Our results revealed that AtGLR3.6 gene was expressed in roots as well as shoots. Moreover, AtGLR3.6 protein was observed in the plasma membrane. Furthermore, the loss-of-function of AtGLR3.6 (glr3.6-1) leads to reduced primary root growth and fewer lateral roots. The over-expression of AtGLR3.6 significantly promoted lateral and primary root and shoot growth in Arabidopsis. The glr3.6-1 mutant exhibited a smaller root meristem size compared with that of the wild-type, indicating that AtGLR3.6 controls root meristem size. In addition, atglr3.6-1 roots show a decreased mitotic activity accounting for reduced root meristem size. The expression of the cyclin-dependent kinase inhibitor Kip-related protein 4 (KRP4) gene was significantly upregulated in the mutant and downregulated in AtGLR3.6-overexpressing roots, suggesting a role of KRP4 in AtGLR3.6-mediated root meristem maintenance. The upregulation of KRP4 in transgenic Arabidopsis exhibited significantly reduced shoot and root growth with reduced root meristem size. Importantly, glr3.6-1 mutant recovered most of its root growth when KRP4 expression was downregulated. However, elevated KRP4 expression in AtGLR3.6-overexpressing plants phenocopied the wild-type root growth. The magnitude of [Ca2+]cyt elevation was reduced in glr3.6-1 mutant roots. Moreover, calcium treatment reduced KRP4 mRNA level, thereby inducing root growth.
To further confirm the role of putative glutamate (glu) signaling, we used a set of compounds that have the ability to modulate the activity of animal iGluRs . We used Glu and other compounds known to agonize or antagonize the animal iGluR channels. iGluR receptor agonists and antagonists altered root development in Arabidopsis. Our results demonstrate that the root growth was promoted by animal iGluR agonists and then was drastically reduced by animal iGluR antagonists. Recovery of root architectures by the addition of Glu in antagonist-treated root is highly related to each other. Taken together, our findings suggest that AtGLR3.6 is a positive regulator that controls lateral and primary root development in Arabidopsis

並列關鍵字

glutamate receptor ； AtGLR ； lateral root ； KRP4 ； auxin ； calcium

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